Title: Chronic alcohol abuse disrupts CD8+ T cell function. In this component we will explore CD8+ T cell functional parameters after chronic ethanol exposure in mice. This work arises from many past observations in chronic human alcoholics who have increased CD8+ T cell activation markers, a shift toward a memory phenotype, and an increased rapid IFNy response after in vitro stimulation. Published work from our laboratories and others has shown comparable findings in CD8+ T cells from mouse spleen, after many weeks of 20% (w/v) ethanol in water ingestion. In recent work we have found that there is a deficient antigen- specific CDS primary response after inoculation with an attenuated strain of the intracellular pathogenic bacterium, Listeria monocytogenes. Additional findings include several subset alterations, reduced expression of the IL-2 receptor beta subunit, and a reduced proliferative rate. Of interest, these changes can be modeled in part by the administration of certain TLR agonists, and the chronic ethanol mice have demonstrable increases in circulating peptidoglycan. In this component we will examine CDS functional responses to CDS immunodominant epitopes and other stimuli. Aim #1 will evaluate CD8+ T cell memory responses to several defined epitopes after inoculation and later challenge of chronic ethanol mice. Recovery of the deficient CD8+ T cell response will be tested after ethanol withdrawal. Direct inoculation of ethanol mice with peptide loaded dendritic cells will be compared with standard inoculation with organisms. Aim #1 tests the hypotheses that a) both primary and memory responses of CD8+ T cells are affected by chronic ethanol;b) that these changes are irreversible;and c) that the defect is not one of antigen presentation in vivo. Aims #2 and #3 will focus on the abnormal signaling responses of CD8+ T cells from chronic ethanol mice, and comparisons with the alterations produced by certain TLR agonists. These aims are based on the observations in CD8+ T cells from chronic ethanol mice and/or TLR agonist treated normal mice, of reduced IL-2R-beta expression, reduced proliferative response of CD8+CD44lo cells and paradoxically increased "expression of activation markers. This component interacts strongly with the Animal Core, the Technical Core, and Research Components #2 &#4 and Pilot Component projects #3 &#4. The PI of Research Components #2 and #4 are collaborators in the experiments of Aim #1. PERFORMANCE SITE(S) (organization, city, state) University of Iowa Carver College of Medicine lowa City, lowa 52242 PHS 398 (Rev. 04/06) Page 166 Form Page 2 Principal Investigator/Program Director (Last. First, Middle): Cook, Robert T., Research Component #1 KEY PERSONNEL. See instructions. Use continuation pages as needed to provide the required information in the format shown below, start with Principal Investigator(s). List all other key personnel in alphabetical order, lasl name first. Name eRA Commons User Name Organization Role on Project Cook, Robert T. COOKRT Colgan, John D. OTHER SIGNIFICANT CONTRIBUTORS Name Organization Zimmerman, M. Bridget University of lowa Legge, Kevin L University of lowa Schlueter, Annette, J. University of lowa University of lowa PI University of lowa Co-I Role on Project Statistician Collaborator 'Collaborator Human Embryonic Stem Cells ^ No Q Yes If the proposed project Involves human embryonic stem cells, liatl>elow the registration numberof the specific cell llne(s)from the following list: http://stemcells.nih.aov/reqistrv/index.asp. Use continuation pages as needed. If a specific line cannot be referenced at this time. Include a statement that one from the Registry will be used. Cell Line PHS 398 (Rev. 04/06) Page 167 Form Page 2-continued Number the folbwing pages consecutively throughput the application. Do not use suffixes such as 4a, 4b. Principallnvestigator/Program Director (Last, First, Middle): Cook. Robert T.. Research Component #1 TABLE OF CONTENTS Page Numbers Research Component #1 Abstract and Key Personnel;. 166-167 Table of Contents 168 1. SpecificAims... : 169 2. Background and Significance 170-173 3. Preliminary Studies;173-180 4. Component Research Design and Methods 180-193 5. Resources and Environment 193 6. Human Subjects N/A 7. Vertebrate Animals 193-195 8. Literature Cited , 195-199 9. Consortium/Contractual Arrangements '. N/A 10. Consultants/Collaborators N/A Appendices on CD: 5 Publications PHS 398 (Rev. 04/06) Page 168 Form Page 3